Aside from being potential precursors to the building blocks of nucleic acids, the natural nucleosides are important metabolites having many physiological effects in assorted organs. For example, adenosine and its corresponding nucleotides are local signaling molecules that act through purinergic receptors to affect such varied physiological functions as lipolysis, neurotransmitter release, coronary vasodilation, cardiac contractility, renal vasoconstriction, and bronchial constriction; and thus extracellular adenosine concentrations can have significant effects on cardiac and vascular functions as well as play a role in neuromodulation [reviewed by Griffith et al. Biochim. Biophys. Acta Rev. Biomembr., 1286:153-181 (1996); Cass, in Drug Transport in Antimicrobial Therapy and Anticancer Therapy (N. H. Georgopapadakou, ed.(Marcel Dekker)), 403-451 (1995)]. In addition, nucleoside analogs are presently employed as anti-retroviral drugs, and as anticancer drugs. Although some extracellular nucleosides can passively permeate the plasma membrane, most participate in some form of protein mediated transport performed by nucleoside transport proteins. Nucleoside transport proteins play an important role in the uptake and efflux of physiological nucleosides used in DNA and RNA synthesis, lipid and glycogen metabolism, and glycoprotein and glycolipid synthesis. Furthermore nucleoside transport proteins mediate the uptake and efflux of a number of antitumor and antiviral nucleoside analogs in cells [Cass, 1995, supra]. Nucleoside transport inhibitors are currently being investigated as modulators of adenosine action in cerebral and cardiac ischemia to provide protection from reperfusion injury [Rongen et al., J. Clin. Invest. 95:658-668 (1995); Parkinson et al., Gen. Pharmacol. 25:1053-1058 (1994)].
The first nucleoside transporters studied functioned as facilitated diffusion systems. Such equilibrative nucleoside transport proteins were initially classified solely by their sensitivity to nitrobenzylmercaptopurineriboside (NBMPR). As the study of these proteins progressed, additional characteristics such as permeant selectivity and tissue distribution have been used to further distinguish these proteins [Griffith and Jarvis, Biochim. Biophys. Acta Rev. Biomembr., 1286:153-181 (1996)]. More recently, sodium-dependent concentrative nucleoside transport proteins have also been identified.
At least five distinct nucleoside transport activities have been identified that differ in their permeant selectivity, sensitivity to inhibitors and distribution in normal tissues and tumors [Griffith and Jarvis, 1996, supra]. Two of these activities exhibit equilibrative mechanisms that mediate both the influx and efflux of nucleosides across the plasma membrane, while the other three activities exhibit concentrative, sodium-dependent mechanisms that under physiological conditions mediate only the influx of nucleosides.
The major equilibrative carrier in most cells, es (equilibrative, sensitive) is highly sensitive to the inhibitor NBMPR, having IC.sub.50. values of 0.1 to 1 nM. A human homolog of this protein (hENT1) has recently been cloned (Griffiths et al., Nature Med. 3:89-93 (1997). It has 10 to 11 predicted membrane spanning regions and has some structural similarities to the equilibrative glucose carriers. It does not however, share sequence homology with the glucose transporter family and appears to represent a new family of membrane transport proteins designated ENT for equilibrative nucleoside transporter.
Many cells also contain a second equilibrative transporter ei (equilibrative, insensitive) that is insensitive to nanomolar concentrations of NBMPR, but can be inhibited by higher (.mu.M) concentrations [Belt, Mol. Pharmacol., 24:479-484 (1983); Plagemann and Wohlheuter, Biochim. Biophys. Acta, 773:39-52 (1984)]. This protein, an NBMPR-insensitive equilibrative nucleoside transport protein (iENTP) has remained elusive. Both of the equilibrative transporters accept a broad range of physiological nucleosides and their cytotoxic and antiviral analogs as permeants, although there appear to be differences in their affinity for some nucleosides [Griffith and Jarvis, 1996, supra].
iENTPs also are present in most tumor cells, although the level of iENTP appears to be variable. The concentration of iENTP in a particular tumor cell is likely to be a major determinant in the ability of that cell to grow following the administration of an es transport inhibitor to block the nucleoside salvage pathway, together with an inhibitor of de novo nucleoside synthesis, such as trimetrexate, methotrexate, and tomudex. The level of iENTP in a tumor cell is also likely to be a determinant of the success of using es inhibitors to block the efflux of cytotoxic and antiviral nucleoside analogs from cells. Under such circumstances, cells with higher concentrations of iENTP will have a higher efflux of cytotoxic and antiviral nucleoside analogs, unless an inhibitor of the iENTP is also administered.
NBMPR and its congeners are the most specific and potent inhibitors of the es transporter currently available. The es transporter has a high-affinity binding site for NBMPR that overlaps at least in part with the substrate binding site [Jarvis, in Adenosine Receptors, D. M. F. Cooper and C. Londos, eds., (New York: Alan R. Liss, Inc.), pp. 113-123 (1988)]. NBMPR binds to this site with a dissociation constant of 0.1 to 1 nM and completely inhibits nucleoside uptake via es at concentrations in the nanomolar range [Paterson and Cass, in Membrane Transport of Antineoplastic Agents, I. D. Goldman, ed., (New York: Pergamon Press), pp. 309-329 (1986); Gati and Paterson, in The red cell membrane: structure, function, and clinical implications, P. Agre and J. C. Parker, eds., (New York: Marcel Decker), pp. 635-661 (1989); Jarvis, 1988, supra; Plagemann et al., Biochim. Biophys. Acta., 969:1-8 (1988)]. At high concentrations (&gt;1 .mu.M), however, NBMPR also inhibits the ei transporter [Paterson et al., Mol. Parmacol., 18:40-44 (1980); Belt, Mol. Pharmacol., 24:479-484 (1983); Plagemann and Wohlheuter, Biochim. Biophys. Acta., 773:39-52 (1984)].
Dipyridamole also binds to the NBMPR-binding site of es [Jarvis, Mol. Pharmacol., 30:659-665 (1986)], but is a less potent inhibitor of es than NBMPR [Plagemann and Wohlheuter, Curr. Topics Membr. Trans., 14:225-330 (1980); Paterson and Cass, 1986, supra; Plagemann and Woffedin, Biochim. Biophys. Acta., 969:1-8 (1988)].
Dipyridamole also inhibits the ei transporter, but its potency against this transporter has been unclear. It has been suggested that the es transporter and the ei transporter are equally sensitive to dipyridamole since the curves for inhibition of nucleoside transport are monophasic in cells that possess both transporters [Jarvis, 1988, supra; Plagemann et al., 1988, supra]. However, recent studies with Ehrlich ascites tumor cells in which the es transporter was blocked by addition of low concentrations of NBMPR, suggest that the ei transporter is significantly less sensitive to dipyridamole than es [Hammond, J. Pharmacol. Exp. Ther., 259:799-807 (1991)].
In addition to the two equilibrative nucleoside transporters there are at least three Na.sup.+ -dependent, concentrative nucleoside transport activities that differ from each other, and from the equilibrative transporters, in their substrate specificity. Two of these, cif and cit (also called N1 and N2), exhibit selectivity for purine and pyrimidine nucleosides respectively [Vijayalakshmi et al., J. Biol. Chem., 263:19419-19423 (1988) and Williams et al., Biochem. J., 264:223-231 (1991)]; while the third, cib (also called N3), has a broader selectivity accepting both purine and pyrimidine nucleosides [Wu et al., J. Biol. Chem., 267:8813-8818 (1992); Huang et al., J. Biol. Chem., 268:20613-20620 (1993)]. All three of the concentrative nucleoside transporters are insensitive to NBMPR and dipyridamole at concentrations up to 10 .mu.M; and under physiological conditions mediate only the influx of nucleoside into cells. These concentrative transport activities have been observed predominantly in normal tissues such as kidney [Le Hir and Dubach et al., Pflugers Arch., 401:58-63 (1984); Williams et al., Biochem. J., 264:223-231 (1989); Williams et al., Biochem. J., 274:27-33 (1991); Le Hir et al., Pflugers Arch., 401:58-63 (1990)] and intestine Schwenk et al., Biochim. Biophys. Acta., 805:370-374 (1984); Vijayalakshmi et al., J. Biol. Chem., 263:19419-19423 (1988); Williams et al., Biochem. J., 274:27-33 (1991), and appear to be the major nucleoside transport activity in the specialized epithelial cells of these tissues [Williams et al., Biochem. J., 274:27-33 (1989); Vijayalakshmi et al., J. Biol. Chem., 263:19419-19423 (1988)]. However, low levels of Na.sup.+ -dependent nucleoside transport have been observed in some tumor cells lines (Lee et al., Biochem. J., 274:85-90 (1991); Belt et al., Mol. Pharmacol., 24:479-484 (1993); Crawford et al., J. Biol. Chem., 265:13730-13734 (1990b); Dagnino et al., Cancer Res., 50:6549-6553 (1990)].
cDNA clones have recently been obtained for two of the concentrative nucleoside transporters. Cass and co-workers have cloned rCNT1 from rat intestine. This cDNA encodes a 71 Kd protein with cit-type transport activity in transient expression studies in Xenopus oocytes [Huang et al., J. Biol. Chem., 269:17757-17760 (1994)] and COS cells [Fang et al., Biochem. J., 317:457-465 (1996)]. The second transporter, rSPNT (rCNT2) was cloned from rat liver and encodes a 72 Kd protein that has cif-type transport activity in expression studies in Xenopus oocytes. The CNT1 and SPNT transporters are 64% identical in their deduced amino acid sequences, and have significant homology with the bacterial nupC nucleoside transporters. They do not, however, have significant homology with any known mammalian proteins, and thus represent a new family of mammalian membrane transporters. It should be noted that rCNT1 and rSPNT do not share homology with SNST [Pajor et al., J. Biol. Chem., 267:3557-3560 (1992)], a member of the sodium-dependent glucose transporter family that has weak nucleoside transport activity when expressed in Xenopus oocytes. It is not yet known whether SNST represents a significant nucleoside transport activity in mammalian cells. The human homolog of CNT1 has recently been cloned [Ritzel et al. Am. J. Physiol. (1997)].
The isolation and cloning of nucleoside transport proteins allows the biochemical characteristics of these transport proteins to be individually investigated and exploited. Such analysis is important for drug development, for example, in which drugs can be more readily designed to inhibit specific transport mechanisms. Unfortunately, heretofore, no NBMPR-insensitive equilibrative transport protein has been isolated or cloned, which has severely hampered analogous studies with this major class of nucleoside transporters.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.